Journal: bioRxiv

Article Title: HIV-1 gp120-induced lysosomal stress responses are controlled by TRPML1 redox sensors

doi: 10.64898/2026.03.02.709165

Figure Lengend Snippet: gp120-induced decreases in endolysosome Fe 2+ and increases in cytosolic Fe 2+ were mediated by endolysosome TRPML1 channels (A, B) Endolysosome Fe 2+ levels were measured with LysoRhoNox-1 by flow cytometry and data were presented as fold changes of mean fluorescence intensity (MFI). gp120-induced decreases in endolysosome Fe 2+ levels were significantly decreased by pre-treatment (1 h) with the TRPML1 inhibitors Ned-19 ( A , 1 μM) and ML-SI1 ( B , 10 μM). (C) Cytosolic Fe 2+ levels were measured with Phen Green FL DA by flow cytometry and data were presented as reciprocals of MFI (1/MFI). gp120-induced increases in cytosolic Fe 2+ levels were significantly decreased by the TRPML1 inhibitors ML-SI1 (10 µM), YM201 (5 µM), and Ned-19 (1 µM). YM201, but not ML-SI1 or Ned-19 significantly decreased basal cytosolic Fe 2+ levels. (D, E) Representative Western blot image ( D ) and quantification of ferritin H (FTH1) protein expression levels showed that gp120-induced increases in protein expression levels of FTH1 were blocked by 1 h treatments with Ned-19 (1 µM, 1 h). (F) Representative immunofluorescence images of TRPML1 (green) and nuclei stained with Hoechst 33342 (blue) in U87MG cells transfected with control shRNA or TRPML1 knockdown (TRPML1 KD); TRPML1 KD significantly decreased protein expression levels of TRPML1 by ∼41%. (G) TRPML1 KD significantly decreased basal cytosolic Fe 2+ levels and significantly decreased gp120-induced increases in cytosolic Fe 2+ levels. (H) TRPML1 KD significantly reduced ferric ammonium citrate (FAC)-induced increases in cytosolic Fe 2+ levels. (I) Pre-treatment (1 h) with the TRPML1 agonist NAADP-AM (1 µM) potentiated gp120-induced increases in cytosolic Fe 2+ levels. Data were shown as means and SEM with individual data points (n = 4-11) included on each bar. Two-way ANOVA with Tukey’s multiple comparison tests were used for statistical analyses. * p <0.05, ** p <0.01, *** p <0.001, **** p < 0.0001

Article Snippet: U87MG cells were transfected with either TRPML1 shRNA plasmid (Santa Cruz, Sc-44519) or control shRNA plasmid (Sc-108060) using Jet prime reagent as per manufacturer’s instructions.

Techniques: Flow Cytometry, Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Transfection, Control, shRNA, Knockdown, Comparison

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Representative WB analysis of ADAM9 and MMP7 target genes (left); cell growth proliferation analysis (right). B) WB analysis of ADAM9 and MMP7 (left), cell growth proliferation (middle) and migration (right). Actin was utilized as internal loading control.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Migration

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative Western blot of A) ADAM9, MMP7 and OPN in normal human melanocytes (NHEM) and in a panel of melanoma cell lines, B) ADAM9 isoforms (left), MMP7 and OPN (middle) and corresponding secreted forms (right) in miR-126&126* versus empty vector-transduced Me665/1 and A375M cell lines. C) Representative Real-time PCR analysis of ADAM9 (left), MMP7 (middle) and OPN (right) mRNAs in the A375M cell line. The unresponsive short isoform of ADAM9 mRNA does not carry miR-126&126* binding sites in its 3′UTR. D) Relative expression values obtained by western blot analysis of ADAM9 (left), MMP7 (middle) and OPN (right) in A375M cells transfected with oligomers mimicking mature miR-126 or miR-126* vs non targeting (no Targ); GAPDH and actin were internal loading controls in RT-PCR and WB, respectively. Columns, of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Binding Assay, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Luciferase reporter assays performed by transfecting a Luc reporter gene (psiCHECK2) linked to 3′-UTR of ADAM9 or MMP7 or OPN or PI3KR2 in miR- versus empty vector-transduced A375M cell lines. B) Schematic presentation of predicted miR-126 and miR-126* target sites identified in the ADAM9 3′UTR (left) and relative miR-126&126*-dependent luciferase activities (right) in presence of wild-type (WT) or mutant (mut) binding sites in the-ADAM9 3′UTR. C) The same schematic representation (left) and luciferase experiments (right) carried out on MMP7 3′UTR. Columns, of minimum of 5 experiments per group; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Western blot analyses showing the effectiveness of stable si-ADAM9, si-MMP7 and scrambled control (SCR) transduction. B) Invasion and migration assays in si-ADAM9- or si-MMP7-infected melanoma cell lines compared with scrambled control. C) Invasion (left) and migration (right) in presence of either ADAM9 or MMP7 recombinant proteins in miR-126&126*-transduced A375M cell lines compared with control cells. Columns, mean±SD of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Transduction, Migration, Infection, Recombinant

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative WB of A) pro-HB-EGF (bottom) and relative densitometric analysis (top) in miR-126&126*- versus empty vector-transduced Me665/1 melanoma cell line treated or not with PMA. B ) pro-HB-EGF and HB-EGF-C levels in si-ADAM9- or si-MMP7-infected melanoma compared with si-scrambled control. C) Real time PCR analysis of ADAM9 and MMP7 in the same silenced cells.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction